CN102247600B - Method for preparing diluent for pseudorabies live vaccine - Google Patents
Method for preparing diluent for pseudorabies live vaccine Download PDFInfo
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- CN102247600B CN102247600B CN 201010181096 CN201010181096A CN102247600B CN 102247600 B CN102247600 B CN 102247600B CN 201010181096 CN201010181096 CN 201010181096 CN 201010181096 A CN201010181096 A CN 201010181096A CN 102247600 B CN102247600 B CN 102247600B
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Abstract
The invention relates to a method for preparing diluent for a pseudorabies live vaccine. The method comprises the following steps of: placing nano immune particle liquid into a reaction container filled with diluent, controlling the concentration of the nano immune particle liquid to be 15wt%, starting a stirring device to stir for 20-40 minutes, controlling the temperature of the reaction container to be 2-8 DEG C, standing for 10-20 hours, and then sterilizing for 10-20 minutes at high temperature of 115 DEG C, thus obtaining the product. Compared with the prior art, the vaccine diluent prepared by the method provided by the invention can improve the immune effect of a pseudorabies vaccine and enhance occupied duplication of pseudorabies virus in lymphocyte and cerebral nerve, thus effectively avoiding latent infection of pseudorabies wild virus, providing guarantee for preventing and eliminating pseudorabies, and controlling establishment and reactivation of PRV (pseudorabies virus) latent infection.
Description
Technical field
The present invention relates to a kind of preparation method of vaccine diluent, especially relate to the preparation method that a kind of pseudorabies live vaccine is used diluent.
Background technology
Pseudorabies virus (Pseudorabies virus, PRV) is a kind of a-herpesvirus, can cause the pseudorabies of multiple domestic animal and wild animal.Other animal except pig shows as lethal infection, and pig is natural host and the reservoir host of PRV, is epidemic infection.Adult Pig usually shows as inapparent infection and respiratory symptom, sow miscarriage, product stillborn fetus and mummy tire, and boar shows as orchitis.Piglet shows heating, stupor and nervous symptoms, and mortality rate is very high.Therefore, pseudorabies is a kind of important breeding difficulty sexually transmitted disease of pig industry.Western developed country all drops into purification and the elimination plan that a large amount of material resources and manpower successively start and finished porcine pseudorabies.In the enforcement of this plan, the latent infection problem of PRV is most important difficulty wherein.
The latent infection of herpesvirus is divided into three phases, i.e. the foundation of latent infection, keep and activate.At establishment stage, virus must enter specific cell, and along with entering, must set up specific viral gene expression to guarantee the pathological change that virus effectively infects not occur.Therefore, many with copy relevant gene, transcribe with function on static.Only have a part of viral gene seldom to express in this period.The viral DNA of latency is not only stable, sets up latent infection but also can suppress other strain.Actually or the function of this mechanism dependovirus needs the auxiliary not clear at present of cytokine, but set up the repeated infection that the attenuated live vaccines strain of latent infection can stop the wild type strain to cause.
Studies show that recently, the negative target tissue that vaccine strain can be transplanted to immune swine is nervi trigeminus unit, and by the superinfection competition malicious with the PR open country, thereby the generation (Schang, 1994) of blocking-up inapparent infection.Vaccine depends on the occupy-place effect of vaccine strain and strain to the barrier effect of wild type strain virus.Lack glycoprotein gE, Gg encoding gene or cause the vaccine of weak gene strain, very weak at the transfer ability of trigeminal ganglion.So currently available vaccines, existing vaccines can not stop the latent infection of the wild poison of pseudorabies.
Latent infection is a kind of special state that virus exists, and is one of common trait of herpetoviridae member.Therefore be in virus long-term existence in animal body of latent infection, but do not produced infective virion, be with malicious animal without any symptom, also toxin expelling not.PRV can not only set up latent infection, and can irregularly be activated and enter the proliferative state, produces poison and discharges, so that become the potential source of infection with malicious animal, cause the infection of other susceptible animal, the control of pseudorabies brought very large difficulty, thus the latent infection of PRV extremely people pay attention to.And vaccine immunity can only stop the appearance of animal morbidity and clinical symptoms, but can not control the foundation of PRV latent infection and reactivate.If eliminate whole PRV latent infection pigs, need to pay huge economy and social costs, do not meet the current national conditions of China.Therefore, the latent infection problem of PRV is the major obstacle of the pseudorabies prevention of current China and elimination plan.
In addition, vaccine immunity can produce certain stress, such as fervescence etc., this also affects the identification with antigen of copying of virus, thereby affects immune effect.
How to strengthen vaccine in nervous system and lymphoid occupy-place effect, be the key that purifies pseudorabies.
Summary of the invention
Purpose of the present invention is exactly to provide a kind of raising immune effect for the defective that overcomes above-mentioned prior art existence, and the enhancing occupy-place copies, avoids the pseudorabies live vaccine of the latent infection preparation method of diluent.
Purpose of the present invention can be achieved through the following technical solutions:
A kind of pseudorabies live vaccine preparation method of diluent, it is characterized in that, the method may further comprise the steps: nano immune particle liquid is placed the reaction vessel that diluent is housed, the concentration of control nano immune particle liquid is 15wt%, open agitating device and stir 20～40min, control reaction vessel temperature is 2～8 ℃, leaves standstill 10～20h, then place 115 ℃ of high temperature sterilize 10～20min, namely obtain product.
Described nano immune particle liquid prepares by following steps: add glucosides Saponin monomer, cholesterol, vitamin C and glu famine in sodium selenite aqueous solution, add Polyethylene Glycol after stirring 5～6min, continue to stir 20～40min, obtain the liquid that golden yellow contains the nano immune particle.
The concentration of sodium selenite is 55～58 μ g/ml in the described sodium selenite aqueous solution, and the content of glucosides Saponin monomer is 0.1～1wt%, and the content of cholesterol is 0.1～1wt%, and the content of vitamin C and glu famine meets following condition:
The weight ratio of sodium selenite and Polyethylene Glycol is 1 in the described sodium selenite aqueous solution: (0.1～1).
Described diluent comprises normal saline or commercially available PBS buffer.
The preferred commercially available PBS buffer of described diluent.
Described agitating device is magnetic stirring apparatus or SANYE blade.
The storage temperature of described product is 0～35 ℃.
Preferred 2～8 ℃ of the storage temperature of described product.
Compared with prior art, the vaccine diluent that the present invention prepares can improve the immune effect of pseudorabies vaccine, strengthening Pseudorabies virus copies in the occupy-place of lymphocyte and cranial nerve, effectively avoided the latent infection of the wild poison of pseudorabies, for prevention and the elimination of pseudorabies provides guarantee, the foundation of control PRV latent infection with reactivate.
Description of drawings
Fig. 1 is Normocellular electromicroscopic photograph;
Fig. 2 is the electromicroscopic photograph after the cytopathy.
The specific embodiment
The present invention is described in detail below in conjunction with specific embodiment.
Embodiment 1
A kind of pseudorabies live vaccine preparation method of diluent, the method may further comprise the steps:
(1) gets the aqueous solution that the 25ml concentration of sodium selenite is 56.4 μ g/ml, stir in lower the adding and add glucosides Saponin monomer, cholesterol, vitamin C and glu famine, wherein the content of glucosides Saponin monomer is 0.1wt%, the content of cholesterol is 0.1wt%, the total mole number of vitamin C and glu famine: the molal quantity of sodium selenite is 2, continue to stir 5min and no longer change rear adding Polyethylene Glycol to solution colour, the weight ratio of Polyethylene Glycol and sodium selenite is 0.1: 1, continue again to stir 20min, obtain the liquid that golden yellow contains the nano immune particle, utilize atomic force microscope to observe, the nano immune particle diameter that obtains is 50nm, in liquid subpackage and the brown receiving flask, be filled with liquid nitrogen and do Preservation in sterile condition, the shelf-life can reach 2 years;
(2) accurate weighing nano immune particle, nano immune particle liquid is placed the reaction vessel that the PBS buffer is housed, the concentration of control nano immune particle liquid is 15wt%, open magnetic stirrer 20min, rotating speed is advisable the most very much not to produce bubble, and control reaction vessel temperature is 2 ℃, leaves standstill 20h and spends the night, then place 115 ℃ of high temperature sterilize 10min, namely obtain product.Being kept in 0 ℃ the environment of the product that obtains, pH is controlled at 6.5.
Embodiment 2
A kind of pseudorabies live vaccine preparation method of diluent, the method may further comprise the steps:
(1) gets the aqueous solution that the 25ml concentration of sodium selenite is 56.4 μ g/ml, stir in lower the adding and add glucosides Saponin monomer, cholesterol, vitamin C and glu famine, wherein the content of glucosides Saponin monomer is 1wt%, the content of cholesterol is 1wt%, the total mole number of vitamin C and glu famine: the molal quantity of sodium selenite is 4, continue to stir 6min and no longer change rear adding Polyethylene Glycol to solution colour, the weight ratio of Polyethylene Glycol and sodium selenite is 1: 1, continue again to stir 40min, obtain the liquid that golden yellow contains the nano immune particle, utilize atomic force microscope to observe, the nano immune particle diameter that obtains is 120nm, in liquid subpackage and the brown receiving flask, be filled with liquid nitrogen and do Preservation in sterile condition, the shelf-life can reach 2 years;
(2) accurate weighing nano immune particle, nano immune particle liquid is placed the reaction vessel that normal saline is housed, the concentration of control nano immune particle liquid is 15wt%, open the SANYE blade and stir 40min, rotating speed is advisable the most very much not to produce bubble, and control reaction vessel temperature is 8 ℃, leaves standstill 10h, then place 115 ℃ of high temperature sterilize 20min, namely obtain product.Being kept in 2 ℃ the environment of the product that obtains, pH is controlled at 7.5.
Embodiment 3
A kind of pseudorabies live vaccine preparation method of diluent, the method may further comprise the steps:
(1) gets the aqueous solution that the 25ml concentration of sodium selenite is 56.4 μ g/ml, stir in lower the adding and add glucosides Saponin monomer, cholesterol, vitamin C and glu famine, wherein the content of glucosides Saponin monomer is 0.5wt%, the content of cholesterol is 0.5wt%, the total mole number of vitamin C and glu famine: the molal quantity of sodium selenite is 3, continue to stir 6min and no longer change rear adding Polyethylene Glycol to solution colour, the weight ratio of Polyethylene Glycol and sodium selenite is 0.5: 1, continue again to stir 40min, obtain the liquid that golden yellow contains the nano immune particle, utilize atomic force microscope to observe, the nano immune particle diameter that obtains is 100nm, in liquid subpackage and the brown receiving flask, be filled with liquid nitrogen and do Preservation in sterile condition, the shelf-life can reach 2 years;
(2) accurate weighing nano immune particle, nano immune particle liquid is placed the reaction vessel that normal saline is housed, the concentration of control nano immune particle liquid is 15wt%, open the SANYE blade and stir 40min, rotating speed is advisable the most very much not to produce bubble, and control reaction vessel temperature is 8 ℃, leaves standstill 10h, then place 115 ℃ of high temperature sterilize 20min, namely obtain product.Being kept in 8 ℃ the environment of the product that obtains, pH is controlled at 7.5.
Embodiment 4
A kind of pseudorabies live vaccine preparation method of diluent, the method may further comprise the steps:
(1) gets the aqueous solution that the 25ml concentration of sodium selenite is 56.4 μ g/ml, stir in lower the adding and add glucosides Saponin monomer, cholesterol, vitamin C and glu famine, wherein the content of glucosides Saponin monomer is 0.5wt%, the content of cholesterol is 0.5wt%, the total mole number of vitamin C and glu famine: the molal quantity of sodium selenite is 3, continue to stir 6min and no longer change rear adding Polyethylene Glycol to solution colour, the weight ratio of Polyethylene Glycol and sodium selenite is 0.8: 1, continue again to stir 40min, obtain the liquid that golden yellow contains the nano immune particle, utilize atomic force microscope to observe, the nano immune particle diameter that obtains is 100nm, in liquid subpackage and the brown receiving flask, be filled with liquid nitrogen and do Preservation in sterile condition, the shelf-life can reach 2 years;
(2) accurate weighing nano immune particle, nano immune particle liquid is placed the reaction vessel that normal saline is housed, the concentration of control nano immune particle liquid is 15wt%, open the SANYE blade and stir 40min, rotating speed is advisable the most very much not to produce bubble, and control reaction vessel temperature is 8 ℃, leaves standstill 10h, then place 115 ℃ of high temperature sterilize 20min, namely obtain product.Being kept in 35 ℃ the environment of the product that obtains, pH is controlled at 7.Table 1 is the stability data of the diluent for preparing.
Table 1
The diluent that utilization prepares is tested the impact of pseudorabies cytotoxic activity.
(1) recovery of VIRO cell, cultivation:
Frozen bhk cell is taken out from liquid nitrogen container, recovery, after growing up to monolayer, with the digestion of 0.25% pancreatin, one EDTA solution, when appropriateness, pour out pancreatin one EDTA solution, add the DMEM culture fluid that contains 10% calf serum, disperse with the piping and druming of sterilization suction pipe, the packing bottle is put in 37 ℃ of incubators and is cultivated.
(2) mensuration of pseudorabies antigen titre:
TCID
50Method: diluent is mixed with PRV (Pseudorabies virus), put under room temperature (25 ℃) environment and place respectively 30min, 2h, be inoculated on the sensitive cells, measure TCID
50Variation.It is matched group that PBS group, DMEM culture fluid group and import PRV vaccine diluent are set.
With Pseudorabies virus with the DMEM culture fluid by 10 times of serial dilutions, namely 10
-1, 10
-2, 10
-3, one 10
-10Deng.The different dilution virus liquids of 100 μ l are added in 96 each hole of porocyte Sptting plate, and each titre is 8 holes, adds subsequently the VIRO cell that 100 μ l have digested dispersion, and cell content is with 1x10
6Cell/mL is advisable, or grows up to monolayer as standard in 24 hours take cell.Put 37 ℃, 5%CO
2Cultivate in the incubator, observe every day, 1 week of Continuous Observation.Record cytopathy hole is pressed Reed-MuenCh Liang Shi method and is calculated TCIDS
50Normal cell as shown in Figure 1, cytopathy is as shown in Figure 2.Table 2 is viral TCID
50Test data.
Table 2
Detect diluent to the research of pig safety, to the diluent for preparing among the pig musculi colli injection 10ml embodiment 1, observe 14 days swinerys and react, the result shows, three kinds of adjuvants all show as good safety, the results are shown in Table 3.
Table 3 large bolus injection safety experiment is table as a result
The diluent that utilizes embodiment 1 to prepare cooperates vaccine that the negative pig of pseudorabies is carried out immunity, detects body temperature in the 24h, and the result of variations of body temperature is as shown in table 4 after the immunity.
Table 4
The diluent for preparing with embodiment 1 cooperates domestic vaccine, PBS to cooperate 5 of each immune pigletss of domestic vaccine, import vaccine and first wife's diluent, uses SABC method research PRV in the field planting at nervi trigeminus and tonsil position behind the 48h.SABC (Streptavidin-Biotin-Peroxidase Complex) method, namely streptavidin-avidin-biotin complex method is a kind of quick tetchy immunohistochemistry staining method.This special-purpose diluent helps the occupy-place of Pseudorabies virus on neural and lymphocyte, helps to prevent inapparent infection.
Claims (4)
1. a pseudorabies live vaccine is with the preparation method of diluent, it is characterized in that, the method may further comprise the steps: nano immune particle liquid is placed the reaction vessel that normal saline or commercially available PBS buffer are housed, the concentration of control nano immune particle liquid is 15wt%, open agitating device and stir 20～40min, control reaction vessel temperature is 2～8 ℃, leaves standstill 10～20h, then place 115 ℃ of high temperature sterilize 10～20min, namely obtain product;
Described nano immune particle liquid prepares by following steps: add glucosides Saponin monomer, cholesterol, vitamin C and glu famine in sodium selenite aqueous solution, add Polyethylene Glycol after stirring 5～6min, continue to stir 20～40min, obtain the liquid that golden yellow contains the nano immune particle;
The concentration of sodium selenite is 55～58 μ g/ml in the described sodium selenite aqueous solution, and the content of glucosides Saponin monomer is 0.1～1wt%, and the content of cholesterol is 0.1～1wt%, and the content of vitamin C and glu famine meets following condition:
The weight ratio of sodium selenite and Polyethylene Glycol is 1 in the described sodium selenite aqueous solution: (0.1～1).
2. a kind of pseudorabies live vaccine according to claim 1 is characterized in that with the preparation method of diluent, and described agitating device is magnetic stirring apparatus or SANYE blade.
3. a kind of pseudorabies live vaccine according to claim 1 is characterized in that with the preparation method of diluent, and the storage temperature of described product is 0～35 ℃.
4. a kind of pseudorabies live vaccine according to claim 1 is characterized in that preferred 2～8 ℃ of the storage temperature of described product with the preparation method of diluent.
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| CN109350738B (en) * | 2018-11-27 | 2022-03-04 | 云南生物制药有限公司 | Pseudorabies live vaccine diluent and preparation method and application thereof |
| CN114344459A (en) * | 2021-12-23 | 2022-04-15 | 河南兴华生物技术有限公司 | Vaccine diluent, preparation method and application thereof |
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| AU557161A (en) * | 1960-06-09 | 1963-05-02 | Thestate Of Bisel | Production of metalliferous coatings |
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